EP2953480B1 - Améliorisation de la biodisponibilité de substances de valeur à partir de microorganismes - Google Patents

Améliorisation de la biodisponibilité de substances de valeur à partir de microorganismes Download PDF

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EP2953480B1
EP2953480B1 EP14702825.2A EP14702825A EP2953480B1 EP 2953480 B1 EP2953480 B1 EP 2953480B1 EP 14702825 A EP14702825 A EP 14702825A EP 2953480 B1 EP2953480 B1 EP 2953480B1
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weight
kwh
cells
cell
process according
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German (de)
English (en)
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EP2953480A1 (fr
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Nicolas Rudinger
Christian Rabe
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Evonik Operations GmbH
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Evonik Operations GmbH
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Priority claimed from EP13153977.7A external-priority patent/EP2762009A1/fr
Priority claimed from DE201310201978 external-priority patent/DE102013201978A1/de
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Priority to EP14702825.2A priority Critical patent/EP2953480B1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C12N1/066—Lysis of microorganisms by physical methods
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment

Definitions

  • the present invention relates to a method for the gentle digestion of cells, foodstuffs and feed containing valuable substances, which contain such valuable substances obtained by gentle digestion, and animals which were obtained by feeding with such feed.
  • omega-3 fatty acids Certain valuable substances, in particular polyunsaturated fatty acids (PUFAs), are an important component for the nutrition of humans and animals.
  • PUFAs polyunsaturated fatty acids
  • fish was primarily used as a source of omega-3 fatty acids. It was later discovered that certain microbes produce heterotrophic omega-3 fatty acids in large quantities, whereby the fatty acid production can be influenced advantageously by choosing specific reaction parameters.
  • the omega-3 fatty acids can then be obtained from the cells or the cells can be used in the form of biomass directly in feed or food.
  • the object of the present task was therefore to provide a gentler process for the production of feed containing valuable substances, preferably lipids, in particular polyunsaturated fatty acids, whereby damage to the valuable substances should be avoided as far as possible.
  • valuable substances preferably lipids, in particular polyunsaturated fatty acids
  • the mechanical energy input can be significantly reduced if the cells are swollen by adding water or an aqueous solution before carrying out the cell disruption process, so that the risk of damage to the valuable substance contained can be greatly minimized.
  • sufficient cell disruption was achieved even with a relatively small amount of water added, which is particularly advantageous with regard to the subsequent further processing.
  • a first subject of the present invention is therefore a method for the disruption of cells which contain valuable substances, in particular lipids, preferably unsaturated fatty acids, characterized in that the cells swell before the cell disruption where, when performing the cell disruption, there is an energy input of 0.1-50 kWh / t on the cell suspension, characterized in that the cells swell by starting from a cell mass with a moisture content of less than 15% by weight. a cell suspension with a moisture content of 30 to 60% by weight is set.
  • “swelling of the cells” is understood to mean that water (or an aqueous solution) penetrates into the cells and thereby the osmotic pressure in the cells is increased.
  • the swelling of the cells takes place according to the invention by starting from a cell mass with a moisture content less than 15% by weight, in particular 1-14% by weight, preferably less than 10% by weight, in particular 1-9% by weight, particularly preferably less than 5% by weight, in particular 1-4.5% by weight, of a cell suspension with a moisture content of 30 to 60 wt .-%, in particular from 35 to 50 wt .-%, is set.
  • the solids content can be determined, for example, using an infrared lamp.
  • the moisture content is preferably determined gravimetrically by determining the water loss during the thermal drying, which can be carried out, for example, in a drying cabinet. Moisture content and solids content are complementary.
  • the cell disruption can be carried out using the cell disruption methods known to the person skilled in the art, such as, for example, by means of a screw extruder, a beater mill, an air jet mill or by using increased pressure, for example by the so-called French press method.
  • the cell disruption is preferably carried out using a rotor-stator system or in an extrusion process.
  • the rotor-stator system is based on a stationary part, called the stator, and a rotating part, the rotor.
  • the rotor typically has a peripheral speed of at least 5 m / s, for example 10 to 30 m / s, the gap width between the rotor and stator can be, for example, 0.1-0.5 mm.
  • the rotor-stator system is preferably a homogenizing system.
  • the MHD 2000 (IKA) mixing machine can be used as the rotor stator, for example.
  • IKA IKA
  • To carry out cell disruption the cell suspension is added to the space between the stator and rotor. The cells are subjected to shear stress in this gap and turbulence also occurs. These two factors cause cell disruption.
  • the energy input to the cells is, according to the invention, 0.1-50 kWh per ton of suspension, the energy input preferably being a maximum of 40, 35 or 30 kWh per ton of suspension, particularly preferably maximum 25, 20 or 15 kWh per ton of suspension.
  • Preferred ranges here are energy inputs of 0.1-50 kWh per ton of suspension, in particular 0.3-45 kWh, particularly preferably 0.5-40 kWh, in particular 0.8-35 kWh, especially 1-30 kWh, in particular 1 , 5 - 25 kWh, 2 - 20 kWh or 3 - 15 kWh, each per ton of suspension.
  • the "cell disruption rate” of the method according to the invention is preferably at least 50%, particularly preferably at least 60, 70 or 80%, especially at least 85, 90 or 95%.
  • the “cell disruption rate” is to be understood as the number of disrupted cells after the cell disruption process has ended in relation to the total number of cells.
  • the cell disruption rate can be determined visually using a microscope as the ratio of the number of disrupted cells to the total number of cells.
  • antioxidants can be contained in the cell suspension used for cell disruption.
  • Preferred antioxidants here are BHT, BHA, TBHA, ethoxyquin, beta-carotene, vitamin E and vitamin C. If used, the antioxidant is preferably present in an amount of 0.01 to 2% by weight.
  • the cell wall-cleaving enzyme is preferably contained in the cell suspension in an amount of 0.01 to 0.5% by weight.
  • the cell disruption is carried out in the absence of enzymes, in particular in the absence of enzymes contribute to the digestion of the cell wall.
  • the enzyme would have to be inactivated again after use.
  • a larger amount of water than preferred according to the invention would be required to effectively use the enzyme.
  • the cells to be used according to the invention in the cell disruption process can be cells which already naturally produce valuable substances, preferably lipids, in particular PUFAs, but can also be cells which have been enabled by appropriate genetic engineering processes to produce lipids, in particular to produce PUFAs.
  • the production can be autotrophic, mixotrophic or heterotrophic.
  • cells are preferably used which produce lipids, in particular PUFAs, heterotrophically.
  • the cells are preferably algae, fungi, in particular yeasts, or protists, but cells from oil-producing plants can also be used, for example.
  • the cells are particularly preferably microbial algae or fungi.
  • the seeds of soybean, flax, rapeseed, corn, cotton, thistle and sunflower come into consideration as cells of oil-producing plants.
  • Strains of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces are particularly suitable as cells of oil-producing yeasts.
  • cells of the taxon Labyrinthulomycetes are preferably used, in particular those of the family Thraustochytriaceae.
  • the genera Althomia, Aplanochytrium, Elnia, Japonochytrium, Schizochytrium, Thraustochytrium and Ulkenia belong to the family of Thraustochytriaceae.
  • cells of the genera Thraustochytrium, Schizochytrium and Ulkenia are particularly preferably used, especially those of the genera Thraustochytrium or Schizochytrium.
  • the strain Schizochytrium limacinum SR21 (IFO 32693) represents a particularly preferred strain.
  • the cells to be used according to the invention are preferably used in the form of so-called "biomass".
  • the biomass is preferably the product of a fermentative cultivation process.
  • the biomass can also contain constituents of the fermentation medium. These components can in particular be salts, anti-foaming agents and unreacted carbon sources and / or nitrogen sources.
  • the cell content in this Biomass is preferably at least 70% by weight, preferably at least 75% by weight.
  • the cell content in the biomass can optionally be increased, for example, to at least 80 or at least 90% by weight by carrying out appropriate washing steps before carrying out the cell disruption process.
  • the biomass obtained can also be used directly in the cell disruption process.
  • the cells to be used according to the invention in the cell disruption process are preferably distinguished by the fact that they have a valuable substance content, preferably lipid content, particularly preferably PUFA content, of at least 20% by weight, preferably at least 30% by weight, in particular at least 40% by weight .-%, each based on the dry cell mass.
  • a valuable substance content preferably lipid content, particularly preferably PUFA content, of at least 20% by weight, preferably at least 30% by weight, in particular at least 40% by weight .-%, each based on the dry cell mass.
  • most of the lipids are in the form of triglycerides, preferably at least 50% by weight, in particular at least 75% by weight and in a particularly preferred embodiment at least 90% by weight of the lipids contained in the cell Form of triglycerides are present.
  • the lipids contained in the cell preferably comprise polyunsaturated fatty acids (PUFAs), preferably at least 10% by weight, in particular at least 20% by weight, particularly preferably 20 to 60% by weight, in particular 20 to 40% by weight , of the fatty acids contained in the cell are PUFAs.
  • PUFAs polyunsaturated fatty acids
  • polyunsaturated fatty acids are understood to mean fatty acids which have at least two C-C double bonds.
  • highly unsaturated fatty acids are preferred among the PUFAs.
  • HUFAs are understood to mean fatty acids which have at least four C-C double bonds.
  • the PUFAs can be present in the cell in free form or in bound form.
  • Examples of the presence in bound form are phospholipids and esters of PUFAs, in particular monoacyl, diacyl and triacylglycerides.
  • a majority of the PUFAs are in the form of triglycerides, preferably at least 50% by weight, in particular at least 75% by weight and in a particularly preferred embodiment at least 90% by weight of the PUFAs contained in the cell Form of triglycerides are present.
  • Preferred PUFAs are omega-3 fatty acids and omega-6 fatty acids, omega-3 fatty acids being particularly preferred.
  • Preferred omega-3 fatty acids are eicosapentaenoic acid (EPA, 20: 5 ⁇ -3), in particular the (5Z, 8Z, 11Z, 14Z, 17Z) eicosa-5,8,11,14,17-pentaenoic acid, and the Docosahexaenoic acid (DHA, 22: 6 ⁇ -3), in particular the (4Z, 7Z, 10Z, 13Z, 16Z, 19Z) -docosa-4,7,10,13,16,19-hexaenoic acid, with docosahexaenoic acid being particularly preferred is.
  • DHA Docosahexaenoic acid
  • the process is preferably carried out as a so-called fed-batch process, ie the carbon and nitrogen sources are supplied incrementally during the fermentation.
  • lipid production can be induced by different measures, for example by limiting the nitrogen source, the carbon source or the oxygen content or combinations thereof.
  • the cells are preferably fermented in a medium with low salinity, in particular to prevent corrosion. This can be achieved by using chlorine-free sodium salts, such as sodium sulfate, sodium carbonate, sodium hydrogen carbonate or soda ash, as the sodium source instead of sodium chloride.
  • Chloride is preferably used in the fermentation in amounts of less than 3 g / l, in particular less than 500 mg / l, particularly preferably less than 100 mg / l.
  • Both alcoholic and non-alcoholic carbon sources can be considered as carbon sources.
  • Examples of alcoholic carbon sources are methanol, ethanol and isopropanol.
  • Examples of non-alcoholic carbon sources are fructose, glucose, sucrose, molasses, starch and corn syrup.
  • inorganic and organic nitrogen sources can be considered as nitrogen sources.
  • inorganic nitrogen sources are nitrates and ammonium salts, especially ammonium sulfate and ammonium hydroxide.
  • organic nitrogen sources are amino acids, especially glutamate, and urea.
  • inorganic or organic phosphorus compounds and / or known growth-stimulating substances such as yeast extract or corn steep liquor, can also be added in order to positively influence the fermentation
  • the cells are preferably fermented at a pH of 4 to 11, in particular 6 to 10, and preferably at a temperature of at least 20 ° C., in particular 20 to 40 ° C., particularly preferably at least 30 ° C.
  • a typical fermentation process takes up to about 100 hours.
  • the cells are harvested after the fermentation has ended.
  • a large part of the fermentation medium can be separated from the biomass by centrifugation, filtration, in particular ultra or microfiltration, decanting and / or solvent evaporation.
  • Solvent evaporation is preferably carried out using a drum dryer, a tunnel dryer, spray drying or vacuum evaporation. Solvent evaporation can in particular also be carried out using a rotary evaporator, a thin-film evaporator or a falling film evaporator.
  • solvent evaporation reverse osmosis to narrow down the fermentation broth can also be used.
  • the biomass obtained is then optionally further dried, preferably by fluid bed granulation. The drying preferably reduces the moisture content to below 15% by weight, in particular to below 10% by weight, particularly preferably to below 5% by weight.
  • the cells are preferably pasteurized after the cells have been harvested or, if appropriate, even shortly before the cells are harvested, in order to kill the cells and inactivate enzymes which could promote the breakdown of the lipids.
  • the biomass obtained in this way - preferably dried - can now be used to produce the cell suspension.
  • Water or an aqueous solution is used to prepare the suspension.
  • the suspension can either be produced directly in the rotor-stator system, so that the suspension and cell disruption are produced in one step, or, alternatively, the cell suspension can first be produced in a stirring system and then used for cell disruption the rotor-stator system.
  • the suspension is produced in the rotor-stator system using a solids-mixing attachment.
  • a solid-mixing attachment is to be understood here as a device which allows the separate introduction of solid on the one hand and water or aqueous solution on the other hand into the rotor-stator system.
  • the suspension is thus only produced during the cell disruption or immediately before the cell disruption by mixing in the solid-mixing attachment.
  • suspensions with very high solids contents can be subjected to cell disruption, which is particularly advantageous with regard to the subsequent processing.
  • Suspensions which are used in the rotor-stator system using a solids-mixing attachment preferably have a solids content of 40-70% by weight, particularly preferably 50-65% by weight.
  • an aqueous solution is used to prepare the cell suspension, then it can in particular contain further food components - such as vitamins or salts.
  • the valuable substance is preferably a lipid, in particular an oil, particularly preferably an oil which contains polyunsaturated fatty acids, preferably at least 10% by weight, in particular at least 20% by weight, particularly preferably 20-60% by weight .-%, especially 20 - 40 wt .-%, of those contained in the cell
  • Lipids represent polyunsaturated fatty acids.
  • Another object of the present invention is a method for producing a food or feed, in which the cells are mixed with other food or feed ingredients after the cell disruption has been carried out and are subsequently processed into the food or feed.
  • the mixture of digested cells and other food or feed ingredients is processed by an extrusion process in order to obtain ready-to-sell portions of the food or feed.
  • a pelleting process can also be used.
  • the undigested cells are first mixed with the other food or feed ingredients, so that the cells are only disrupted during the extrusion process.
  • Another object of the present invention is therefore also a process for the production of a food or feed, in which the cells of the cell suspension are mixed with other food or feed ingredients before the cell disruption is carried out, and the mixture thus obtained is extruded into a food or feed is processed.
  • a screw or twin-screw extruder is preferably used in the extrusion process.
  • the extrusion process is preferably carried out at a temperature of 80-220 ° C, in particular 100-190 ° C, a pressure of 10-40 bar, and a shaft rotation speed of 100-1000 rpm, in particular 300-700 rpm.
  • the residence time of the introduced mixture is preferably 5 to 30 seconds, in particular 10 to 20 seconds.
  • the method comprises a compacting and a compression step.
  • the components are preferably intimately mixed with one another. This is preferably done in a drum equipped with blades. In a preferred embodiment, this mixing step is carried out by water vapor injection, in particular in order to cause the starch preferably contained to swell.
  • the other food or feed ingredients are preferably comminuted - if necessary - before mixing with the disrupted or non-disrupted cells to ensure that a homogeneous mixture is obtained in the mixing step.
  • the other food or feed ingredients can be crushed, for example, using a hammer mill.
  • Processes according to the invention for producing a food or feed are preferably distinguished by the fact that in no process step is there a higher energy input to the cells or disrupted cells than in cell disruption.
  • the energy input in all other process steps to which the cells or disrupted cells or the valuable substance contained are exposed is preferably significantly lower than during cell disruption.
  • the energy input in all other method steps is preferably a maximum of 80%, in particular a maximum of 60%, of the energy input which takes place on the cells during cell disruption. This ensures that the recyclable material contained is damaged as little as possible.
  • the disrupted cells preferably make up 0.5-20% by weight, in particular 1-10% by weight, preferably 2-8% by weight, of the food or feed or of the composition used to produce the food or feed .
  • the present invention also relates to food or feed which can be obtained by the process according to the invention.
  • the foodstuffs and feedstuffs obtainable by the process according to the invention described above are preferably distinguished by a very homogeneous distribution of the valuable substance in the available foodstuff or animal feedstuff.
  • the valuable substance here preferably makes up 0.1-15% by weight, particularly preferably 0 , 2 - 8% by weight of the food or feed.
  • the homogeneity of the distribution can be determined in the following way: n samples each with a mass of x grams are taken. Then the mass of the valuable substance (y grams) contained in the individual samples is determined and the mean value of the mass of the valuable substance contained in the n samples is determined (y n grams).
  • Food or feed obtained by the method according to the invention are preferably characterized in that the deviation of the contained mass of the valuable substance in the individual samples with respect to the determined mean value of the mass of the valuable substance is at most 25%, preferably at most 20 or 10%, particularly preferably at most 5, 3 or 2%.
  • This feature is preferably fulfilled for at least one of the following specifications, preferably for all of the following specifications: 20 or 50 samples of the food or feed with a mass of 1.00 gram each, 20 or 50 samples of the food or feed with a Mass of 500 milligrams each, 20 or 50 samples of the food or feed with a mass of 100 milligrams each.
  • the variance in the distribution of the valuable substance in different portions of the food or feed is at most 10%, preferably at most 5%, in particular at most 3%.
  • the variance is preferably also determined here for 20 or 50 samples of the food or feed with a mass of 1.00 gram, 500 milligrams or 100 milligrams, the characteristic of the variance preferably for at least one of the specifications, particularly preferably for all of the above Requirements is met.
  • the food or feed is preferably an agent for use in aquaculture or a food or feed for use in poultry, pig or cattle breeding.
  • the feed can also be a feed that is used to breed small organisms that can be used as feed in aquaculture. Small organisms can be, for example, nematodes, crustaceans or rotifers.
  • the feed is preferably flake, spherical or tablet-shaped.
  • a feed obtainable by extrusion preferably has a moisture content of less than 5% by weight, particularly preferably from 0.2 to 4% by weight.
  • the other food or feed ingredients are preferably selected from protein-containing, carbohydrate-containing, nucleic acid-containing and lipid-soluble components and, if appropriate, further fatty components and further from other additives such as minerals, vitamins, pigments and amino acids.
  • it can also contain structuring substances to improve the texture or appearance of the feed.
  • binders can also be used, for example, to influence the consistency of the feed.
  • a preferred component that is both a nutritional substance and a structuring substance is starch.
  • Fish protein, krill meal, mussel meal, squid meal or shrimp shells can be used, for example, as a protein-containing component which additionally contains fats.
  • Fish oil can alternatively be used as a fatty component.
  • a vegetable oil can also be used as the fatty component, in particular oil from soybeans, rapeseed, sunflower seeds and flax seeds.
  • wheat flour, sunflower flour, soybean meal or cereal gluten can be used as the carbohydrate-containing component.
  • the total content of oil in the feed - including the oil from the oil-containing cells - is preferably 15 to 40% by weight, in particular 15 to 30% by weight.
  • the feed for use in aquaculture is preferably used to breed fin fish and crustaceans, which are preferably used for human nutrition. These include carp, tilapia, catfish, tuna, salmon, trout, baramundi, bream, perch, cod, shrimp, lobster, crab, shrimp and crayfish. It is particularly preferably a feed for salmon farming.
  • Preferred types of salmon are the Atlantic salmon, the sockeye salmon, the masu salmon, the king salmon, the ketal salmon, the silver salmon, the Danube salmon, the Pacific salmon and the humpback salmon.
  • fish meal or fish oil can in turn be used in aquaculture to breed edible fish or crustaceans.
  • Aquaculture can take place in ponds, tanks, basins or even in delimited areas in the sea or in lakes, in particular in cages or net pens. Aquaculture can be used to breed the finished edible fish, but can also be used to breed juvenile fish, which are then released to add to the wild fish population.
  • Example 1 Production of a biomass containing DHA
  • the strain Schizochytrium limacinum SR21 was used to produce DHA. This is deposited with the NIBH under FERM BP-5034 and with the IFO under IFO 32693.
  • the S. limacinum SR21 strain was originally isolated from sea water ( Nakahara et al. 1996, JAOCS, 73 (10 ); Honda Mycol. Res. 1998 ).
  • the strain was fermented in a medium which contained 50% artificial sea water (Sigma Aldrich) and also the following components: 60 g / l glucose, 0.7 g / l corn steep liquor (Sigma Aldrich), 2 g / l (NH 4 ) 2 SO 4 and 3 g / l KH 2 PO 4 .
  • the fermentation was carried out at 28 ° C., a pH of 4.0, an aeration rate of 0.5 vvm and a stirring of 200 rpm for 60 hours. After the end of the fermentation, an antioxidant was added to the fermentation broth and the fermentation broth was then heated to 60 ° C. for at least 20 minutes.
  • the biomass was then dried in two stages: first, the fermentation broth was evaporated to a dry matter of about 20% by weight.
  • the concentrated fermentation broth was then spray dried using a Production Minor TM Spray Dryer (GEA NIRO) at an inlet temperature of the drying air of 340 ° C. Spray drying gave a powder with a dry matter of more than 95% by weight.
  • GSA NIRO Production Minor TM Spray Dryer
  • the dried biomass was disrupted using a MHD 2000 rotor-stator system (IKA, Germany). Due to a solids-mixing attachment, this rotor-stator system allows the cell suspension to be made available only immediately before the cell disruption. I.e. the dry cell mass and water are mixed in situ shortly before or during homogenization.
  • Example 1 For this purpose, the cell powder from Example 1 was added to the solid-mixing attachment from above and water was metered in laterally to swell the cells in order to hereby to provide a suspension with a dry matter content of 55% by weight or 60% by weight.
  • the cell suspension thus produced in situ is immediately homogenized by the rotor stator.
  • a hexane extraction was then carried out with the homogenized cell suspension in order to determine the content of freely available and thus extracellular polyunsaturated fatty acid DHA.
  • a hexane extraction was carried out with the undigested biomass.
  • the cell disruption was also analyzed optically using a scanning electron microscope.
  • the total DHA content in the cells was determined by a flame ionization detector (FID).
  • FID flame ionization detector
  • the cells of a sample were disrupted, saponified with potassium hydroxide and then acidified using hydrochloric acid.
  • the free fatty acids were then methylated using BF 3 (boron trifluoride 30% in methanol) and separated by means of distribution chromatography with a temperature gradient.
  • each 250 mg sample was weighed into 10 ml pyrex tubes (Pyrex) and 1 ml of internal standard - prepared in hexane - was added. Then 5 ml of hexane was added and the sample was shaken overnight. Then 1 ml of the sample obtained in this way was pipetted into a 10 ml pyrex tube and evaporated in a thermoblock at 50 ° C. while gassing with nitrogen. 2 ml of 0.5 N KOH were added to the drying residue obtained in this way, the tube was tightly closed and the mixture obtained was incubated for 15 min at 100 ° C.
  • the swelling of the cells thus represents an effective means of enabling the cells to be disrupted with a low energy input.
  • the relatively low water input before cell disruption also ensures that the product obtained can be processed into a feed without further concentration

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Claims (10)

  1. Procédé de désintégration de cellules microbiennes, qui contiennent une substance de valeur, caractérisé en ce que les cellules sont gonflées avant la désintégration cellulaire, un apport d'énergie à la suspension cellulaire de 0,1 à 50 kWh/t ayant lieu lors de la réalisation de la désintégration cellulaire, caractérisé en ce que le gonflement des cellules a lieu en ce qu'à partir d'une masse cellulaire ayant une teneur en humidité de moins 15 % en poids, une suspension cellulaire ayant une teneur en humidité de 30 à 60 % en poids est ajustée.
  2. Procédé selon la revendication 1, caractérisé en ce que la substance de valeur consiste en un lipide, le lipide représentant de préférence au moins 20 % en poids de la masse cellulaire et contenant de préférence des acides gras polyinsaturés.
  3. Procédé selon la revendication 1 ou 2, caractérisé en ce que le gonflement des cellules a lieu en ce qu'à partir d'une masse cellulaire ayant une teneur en humidité de moins de 15 % en poids, de préférence de moins de 10 % en poids, notamment de moins de 5 % en poids, une suspension cellulaire ayant une teneur en humidité de 30 à 60 % en poids, avant tout de 35 à 50 % en poids, est ajustée.
  4. Procédé selon l'une quelconque des revendications précédentes, caractérisé en ce qu'un système rotor-stator est utilisé pour la réalisation de la désintégration cellulaire.
  5. Procédé selon l'une quelconque des revendications 1 à 3, caractérisé en ce que la désintégration cellulaire a lieu dans un procédé d'extrusion.
  6. Procédé selon la revendication 5, caractérisé en ce que les cellules sont mélangées avant la réalisation du procédé d'extrusion avec d'autres constituants d'aliments ou d'aliments pour animaux, et transformées en un aliment ou aliment pour animaux dans le procédé d'extrusion.
  7. Procédé selon l'une quelconque des revendications précédentes, caractérisé en ce qu'un apport d'énergie à la suspension cellulaire de 0,5 à 40 kWh/t, notamment de 1 à 30 kWh/t, avant tout de 3 à 15 kWh/t, a lieu lors de la réalisation de la désintégration cellulaire.
  8. Procédé selon l'une quelconque des revendications précédentes, caractérisé en ce que les cellules microbiennes consistent en des labyrinthulomycètes, de préférence de la famille des Thraustochytriaceae, de manière particulièrement préférée des genres Thraustochytrium, Schizochytrium ou Ulkenia.
  9. Procédé selon l'une quelconque des revendications précédentes, caractérisé en ce que les cellules représentent 0,5 à 20 % en poids, notamment 1 à 10 % en poids, de préférence 2 à 8 % en poids, de la composition utilisée dans le procédé d'extrusion, et les autres constituants d'aliments ou d'aliments pour animaux sont choisis parmi les composants contenant des glucides, contenant des protéines, contenant des acides nucléiques, solubles dans les lipides et éventuellement d'autres composants contenant des matières grasses.
  10. Procédé de fabrication d'un aliment ou aliment pour animaux, comprenant les étapes suivantes :
    a) la préparation d'une suspension cellulaire de cellules de l'ordre des Thraustochytriales, notamment du genre Thraustochytrium ou Schizochytrium, ayant une teneur en humidité de 30 à 60 % en poids, notamment de 35 à 50 % en poids, à partir d'une masse cellulaire ayant une teneur en humidité de moins de 15 % en poids, de préférence de moins de 10 % en poids, notamment de 1 à 9 % en poids, de manière particulièrement préférée de moins de 5 % en poids, notamment de 1 à 4,5 % en poids,
    b) la désintégration cellulaire en utilisant un apport d'énergie de 0,1 à 50 kWh, notamment 0,3 à 45 kWh, de manière particulièrement préférée 0,5 à 40 kWh, notamment 0,8 à 35 kWh, avant tout 1 à 30 kWh, notamment 1,5 à 25 kWh, 2 à 20 kWh ou 3 à 15 kWh, à chaque fois par tonne de suspension, de préférence en utilisant un système rotor-stator ;
    c) le mélange des cellules désintégrées et/ou de substances de valeur isolées à partir de celles-ci avec d'autres constituants d'aliments ou d'aliments pour animaux ;
    d) la fabrication du produit final par un procédé de compactage ou d'extrusion.
EP14702825.2A 2013-02-05 2014-02-03 Améliorisation de la biodisponibilité de substances de valeur à partir de microorganismes Active EP2953480B1 (fr)

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EP13153977.7A EP2762009A1 (fr) 2013-02-05 2013-02-05 Améliorisation de la biodisponibilité de substances de valeur à partir de microorganismes
DE201310201978 DE102013201978A1 (de) 2013-02-07 2013-02-07 Verbesserte Bioverfügbarkeit von Wertstoffen aus Mikroorganismen
EP14702825.2A EP2953480B1 (fr) 2013-02-05 2014-02-03 Améliorisation de la biodisponibilité de substances de valeur à partir de microorganismes
PCT/EP2014/052034 WO2014122092A1 (fr) 2013-02-05 2014-02-03 Amélioration de la biodisponibilité de substances de valeur issues de micro-organismes

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Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2826384A1 (fr) 2013-07-16 2015-01-21 Evonik Industries AG Procédé destiné au séchage de biomasse
CA2958457C (fr) 2014-10-02 2022-10-25 Evonik Industries Ag Procede de production d'une biomasse contenant des agpi qui presente une haute stabilite cellulaire
BR112017006834B1 (pt) * 2014-10-02 2022-04-26 Evonik Operations Gmbh Processo para a preparação de um alimento para animais compreendendo pufas, produto extrudado de alimento para animais e método de criação de animais
BR112017005388B1 (pt) 2014-10-02 2022-09-13 Evonik Operations Gmbh Alimento para animais contendo biomassa de aurantiochytrium
EP3200603A1 (fr) 2014-10-02 2017-08-09 Evonik Degussa GmbH Aliment pour animaux contenant des acides gras polyinsaturés, à haute résistance à l'abrasion et à grande hydrostabilité
US11419350B2 (en) 2016-07-01 2022-08-23 Corbion Biotech, Inc. Feed ingredients comprising lysed microbial cells
BR112019000435A2 (pt) 2016-07-13 2019-04-30 Evonik Degussa Gmbh método para separar lipídios de uma biomassa contendo lipídios lisados
US11352651B2 (en) 2016-12-27 2022-06-07 Evonik Operations Gmbh Method of isolating lipids from a lipids containing biomass
EP3470502A1 (fr) 2017-10-13 2019-04-17 Evonik Degussa GmbH Procédé de séparation des lipides à partir de biomasse contenant des lipides lysés
EP3527664A1 (fr) 2018-02-15 2019-08-21 Evonik Degussa GmbH Procédé d'isolement de lipides à partir de biomasse contenant des lipides
AU2018102207A4 (en) * 2018-04-04 2021-02-04 XIAMEN HUISON BIOTECH Co.,Ltd. Application of schizochytrium limacinum and preparation thereof in improvement of quality and yield of animal product
WO2019219396A1 (fr) 2018-05-15 2019-11-21 Evonik Operations Gmbh Procédé d'isolement de lipides à partir de biomasse contenant des lipides par inversion d'émulsion
US11414621B2 (en) 2018-05-15 2022-08-16 Evonik Operations Gmbh Method of isolating lipids from a lipids containing biomass with aid of hydrophobic silica
FR3085825B1 (fr) 2018-09-14 2021-07-16 Fermentalg Huile de microorganismes riche en acide docosahexaenoique

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120183668A1 (en) * 2010-08-11 2012-07-19 E.I. Du Pont De Nemours And Company Aquaculture feed compositions

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5340742A (en) 1988-09-07 1994-08-23 Omegatech Inc. Process for growing thraustochytrium and schizochytrium using non-chloride salts to produce a microfloral biomass having omega-3-highly unsaturated fatty acids
US6977167B2 (en) * 1988-09-07 2005-12-20 Martek Biosciences Corporation Mixtures of omega-3 and omega-6 highly unsaturated fatty acids from euryhaline microorganisms
US5130242A (en) 1988-09-07 1992-07-14 Phycotech, Inc. Process for the heterotrophic production of microbial products with high concentrations of omega-3 highly unsaturated fatty acids
CN101928213B (zh) 1996-03-28 2012-12-05 Dsmip资产有限公司 粒状微生物生物质的制备及其有价值的化合物的分离方法
CN1217029A (zh) 1996-03-28 1999-05-19 吉斯特-布罗卡迪斯股份有限公司 从用巴氏法灭菌的生物量中制备含有微生物多不饱和脂肪酸的油的方法
DE60143287D1 (de) * 2000-01-19 2010-12-02 Martek Biosciences Corp Lösungsmittelfreies extraktionsverfahren
CA2396691C (fr) 2000-01-28 2012-09-18 Omegatech, Inc. Production amelioree de lipides contenant des acides gras polyenes au moyen de cultures a grande densite de microbes eucaryotes dans des fermenteurs
EP1924290A4 (fr) * 2005-05-12 2011-05-25 Martek Biosciences Corp Hydrolysat de biomasse, ses utilisations, et sa production
US20110045564A1 (en) * 2009-08-20 2011-02-24 Srisuda Dhamwichukorn Method of enhanced sustainable production of algal bio-products, comprising use of symbiotic diazotroph-attenuated stress co-cultivation
EP2384647A1 (fr) * 2010-05-07 2011-11-09 Adexgo Ltd. Compositions d'additifs alimentaires et leur procédé de production

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120183668A1 (en) * 2010-08-11 2012-07-19 E.I. Du Pont De Nemours And Company Aquaculture feed compositions

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